PROJECT SUMMARY In the heart, KCNQ1 voltage-gated potassium channels form the alpha-subunits for the slow delayed rectifier potassium current, a repolarizing current critical for cardiac action potential termination. Calmodulin (CaM) is an obligatory KCNQ1 auxiliary subunit which binds the KCNQ1 cytoplasmic domain and regulates normal KCNQ1 trafficking and function. Inherited mutations in KCNQ1 (>600) and CaM lead to congenital long QT syndrome (LQTS), which predisposes patients to arrhythmias and sudden cardiac death. Little is known regarding the specific KCNQ1 mutations or the molecular mechanism underlying CaM dysregulation of KCNQ1 to cause LQTS. Conversely, because CaM binds multiple ion channel targets in cardiomyocytes, it is unclear whether CaM mutations cause LQTS by inducing KCNQ1 dysfunction. This study?s overall goal is to elucidate mechanisms of CaM regulation of KCNQ1 and identify arrhythmogenic mutations in KCNQ1 and CaM which cause LQTS specifically through CaM dysregulation. Until recently, the lack of KCNQ1 structural and gating mechanism insights has hindered mechanistic studies of CaM regulation of KCNQ1. However, recent breakthroughs in both functional and structural studies of KCNQ1 revealed novel findings which may overcome this barrier. This proposal is motivated by three novel CaM regulatory hypotheses derived from these studies: (1) CaM regulates KCNQ1 through simultaneous interactions with the KCNQ1 cytoplasmic and transmembrane domains, (2) CaM exerts selective regulation on distinct KCNQ1 open states, and (3) CaM regulation requires concurrent ATP binding. Presently, there is little functional data to support these hypotheses as genuine gating mechanisms. This proposal plans to investigate these novel CaM regulatory hypotheses with three aims. Aim 1 will characterize the functional impact of disrupting CaM simultaneous interactions with the transmembrane and cytoplasmic domains with systematic mutagenesis, electrophysiological recordings, and optical assays. Aim 2 will utilize a biophysical approach to probe the mechanistic hypothesis that CaM alters KCNQ1 current through selective regulation of distinct KCNQ1 open states with mutagenesis and protein engineering methods. Aim 3 will expand the scope to multi-ligand KCNQ1 regulation. Aim 3 will answer whether CaM regulation of KCNQ1 requires concurrent ATP binding to the KCNQ1 cytoplasmic domain with combined electrophysiology and fluorescence techniques. Results from this study will functionally establish novel mechanisms of CaM regulation of KCNQ1, identify which and how mutations in KCNQ1 and CaM lead to LQTS due to CaM dysregulation. These findings will pave the way for a precision medicine approach to congenital LQTS and may reveal therapeutic approaches against LQTS through targeting these novel CaM regulatory mechanisms.